BRPI0614917B1 - PROCESS FOR OBTAINING A NATURAL CONCENTRATE RICH IN NATURAL HYDROXYROSROS FROM WASTE OF OLIVEIRA AND BY-PRODUCTS - Google Patents

Abstract

process for obtaining a natural concentrate rich in hydroxytyrosol from olive residues and by-products using clean technologies. The present invention relates to a process for obtaining a natural, bioactive, hydroxytyrosol-rich concentrate from olive residues and by-products using clean technologies. These technologies include supercritical fluid extraction, nanofiltration and reverse osmosis, which are used individually or in an integrated mode. The natural extract comprises a minimum concentration of 15% (mass fraction) of hydroxytyrosol and a maximum concentration of 98% (mass fraction) of this compound. The hydroxytyrosol-rich concentrate exhibits antioxidant, antimicrobial, anti-inflammatory and anticarcinogenic activities, which are superior to the activities observed for hydroxytyrosol alone, in equivalent concentration, the hydroxytyrosol-rich concentrate can be prepared as solid particles as a aqueous solution, in emulsion or as lipid based nanoparticles. Industrial application comprises the food, pharmaceutical and cosmetic industries.

Description

PROCESS FOR OBTAINING A NATURAL CONCENTRATE RICH IN NATURAL HYDROXYTYROSOL FROM OLIVE WASTE AND BY-PRODUCTS

Object and field of the invention

The present invention relates to a process for obtaining a natural, bioactive concentrate, from olive residues and by-products, using clean technologies. These technologies include extraction with supercritical fluids, nanofiltration and reverse osmosis. In particular, the invention provides an olive extract containing hydroxytyrosol, and a process for obtaining it.

The hydroxytyrosol-rich concentrate has an important added value as an antioxidant, antimicrobial, anti-inflammatory and anticarcinogenic. The industrial application comprises the food, pharmaceutical and cosmetics industries.

Background of the invention

Over the past few years, some epidemic studies have been correlating the Mediterranean diet with the low frequency of heart disease, atherosclerosis and defined types of cancer. A particular characteristic of the Mediterranean diet is the use of olive oil, consumed directly or used in cooking (Visioli et al., 2002;

Owen et al., 2000).

Studies concerning biphenolic compounds present in olives and olive leaves have led researchers to recognize their biological properties, which were also associated with the healthy positive properties of olive oil.

Per

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2/25 therefore, the bioactive compounds present in olives and olive oil have been recognized as important targets for the pharmaceutical and food industries (Schieber et al., 2001). However, 98% of these biphenolic compounds were lost during the production of olive oil and remained in the vegetation water and / or in the solid residues resulting from the processing of the olives (Rodis et al., 2002).

The compounds present in high concentrations in the solid residues are glycosylated secairidoids, whereas in vegetation water the compounds with higher concentrations are derived from secairides, mainly hydroxytyrosol and oleoeuropein (Mullinacci et al., 2001).

The properties of solid residues obtained during the production of olive oil were studied, as well as the extracts obtained from them (Visioli et al., 1999); in particular, its antibacterial activity has been demonstrated (Ramos-Cormenzana et al., 1996), which has been associated with the presence of oleoeuropein and hydroxytyrosol; the latter compound was highlighted as being the compound with the highest bioactivity (Bisignano et al., 1999).

Hydroxytyrosol was also identified as a potent chemopreventive agent (Manna et al., 2000), and was considered as the component present in olive oil residues with the highest anti-oxidant power. The first recognized properties of hydroxytyrosol were its ability to prevent the oxidation of low-density lipoprotein (LDL) (Visioli and Galli, 1998) and the aggregation of blood platelets (Petroni et al., 1995). Mana et al.

(2000) demonstrated that this compound is able to protect

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3/25 various human cellular systems of toxicity induced by reactive oxygenated species. The ability of hydroxytyrosol to induce DNA changes was also investigated (Aruoma et al., 1999 and Deima et al., 1999).

Visioli et al. (2000) also demonstrated that, depending on the dosage, this biphenolic compound is well absorbed by humans, being excreted in the urine as conjugated glucuronates.

Currently, olive oil is produced exclusively using mechanical and physical processes, which consist of pressing the fruit (pulp and stones) until obtaining a homogeneous paste, which is then processed by phase separation.

The step of separating hydraulic presses, which continuous centrifugation. The so-called three-phase process traditional uses have been replaced by a continuous process and also phases, in which olive oil, vegetation water and olive pomace are obtained as final products, or as a two-phase process, in which the final product streams are olive oil and olive pomace.

In the three-phase system, water is added to the olive paste and this mixture is then treated in a horizontal centrifuge, in which the solid phase is separated from the oily wort. This must is then processed by a vertical centrifuge, in which the oil is separated from the vegetation water.

The most common process today is the two-stage process, as it involves less water consumption. Consequently, it produces a smaller amount of residual water. This process uses two-stage centrifuges, which

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4/25 separate the olive oil and olive pomace. Olive pomace is a semi-solid residue, with the appearance of a mud.

Several patents have been published, which present methods for the recovery of phenolic compounds from the residues of the olive tree. U.S. Patent No.

361 803 describes a process for the recovery of antioxidant compounds from olive residues. This process consists of a preliminary extraction with an aqueous solvent, the produced extract being used to feed an adsorption column, in order to retain the compounds of interest. These compounds are subsequently recovered by eluting an organic solvent through the adsorption column. U.S. Patent No. 6,849,770 describes a process for recovering hydroxytyrosol through a chromatographic process that uses methanol or water / ethanol mixtures as elution solvents.

WO 0218310 describes a process for obtaining a hydroxytyrosol-rich composition from vegetation water, using a previously patented method (U.S. Patent No. 5,490,884), known as Porocrit. The extraction of the desired compounds from the vegetation water is achieved using supercritical fluids, such as carbon dioxide, and porous membranes instead of contact columns. Instead of dispersing the phases, the liquid is fed continuously through porous polypropylene membranes, configured in the form of bundles of hollow fibers or spiral-wound sheets. The liquid passes through the porous membranes inside a pressurized module, while the carbon dioxide

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5/25 supercritical flow counter-current across the membrane. It is important to note, according to the authors of this patent, that the pressure in the module is essentially the same, so that the extraction is caused by the concentration gradient between the liquid and the supercritical carbon dioxide, and not by a pressure gradient. between the two sides of the membrane. The extract can be recovered by vaporizing carbon dioxide for recycling. In addition, U.S. Patent No. 5,714,150 describes a process for extracting oleuropein from olive leaves using water / ethanol mixtures.

The use of filtration processes through membranes and their integration with other techniques, such as centrifugation, has been reported by research groups, with a view to developing processes for the treatment of wastewater from olive mills.

The process shown in Document WO 0218310 differs from the process of the present invention mainly because it does not include a reverse osmosis operation, this operation being comprised in the second step of the present invention. Another aspect that distinguishes this process is the fact that the pressure, on the two opposite sides of the membrane, is essentially the same, so that the extraction is caused by the concentration gradient between the liquid and the supercritical carbon dioxide and not by a gradient pressure between the two sides of the membrane. Consequently, the product obtained by the process described in WO 0218310 is substantially different from the extract obtained according to the process of the present invention, which, most preferably, excludes compounds with a molecular weight

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6/25 above 300 Da.

Drouiche et al. (2004) describe the use of ultrafiltration to remove particles and organic compounds from vegetation water. The work published by 5 Turano et al. (2002) describes a process that integrates centrifugation and ultrafiltration for the treatment of vegetation water. In this integrated process, centrifugation allows the fraction of suspended solids to be removed, protecting the ultrafiltration membrane from serious obstruction problems 10 caused by these particles.

DellaGreca et al. (2001 and 2004) report an analytical method developed for the characterization of vegetation water. This method involves the fractionation of vegetation water, in order to obtain individual isolated components present in the water. A number of membrane techniques are used for this purpose.

US 2003/0108651 refers to a process different from the process described in the present invention, namely because it does not include a reverse osmosis operation, which is included in the second step of the present invention. As a consequence of using different processes, the complex extract obtained according to the process of the present invention is different from the product obtained by the process described in Document US 2003/0108651, which 25 describes that the method comprises the fractionation of water from incubated vegetation, to separate hydroxytyrosol from other components. In addition, this process uses, for the fractionation and separation of hydroxytyrosol, the incubation of vegetation water with an organic solvent, namely ethyl acetate, and also comprises the purification of

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7/25

hydroxytyrosol per chromatography. On the contrary, The present invention no comprises the break up of hydroxytyrosol nor yours purification by chromatography or by any other middle, once your purpose is

obtain a biological extract, which comprises hydroxytyrosol and other bioactive compounds.

EP 1 623 960 A1 describes a process for treating wastewater from olive mills, allowing the recovery of highly pure tyrosol and hydroxytyrosol. In order to obtain said product, this process uses membrane techniques. In addition, the tyrosol compound recovered from wastewater from olive mills is catalytically converted to hydroxytyrosol.

Therefore, this process aims to obtain highly pure compounds, instead of complex natural extracts of olive oil by-products.

Skaltsounis, L. et al. (MINOS PROJECT Development of a process for an integrated management of olive mill waste, recovering natural antioxidants and producing organic fertilizer) describes an adsorption process, since it uses a resin to capture polyphenols from the wastewater of olive mills. The polyphenols captured in the resin medium are therefore recovered using an organic solvent. In addition, these polyphenols are thermally recovered from the organic solvent used and separated by chromatography.

Nanofiltration / reverse osmosis are referred to in this project as a way to treat the resin and brine discharge effluent, which is rich in mineral salts (counter-ions of the resin material), but not

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8/25 contains polyphenols. Thus, the process mentioned above is completely different from the process described in the present invention, due to the use of that resin to isolate the polyphenols from the wastewater of olive mills, instead of the use of a two-step process, which consists in the extraction of the compounds using a supercritical fluid extraction column or a nanofiltration unit, and then use a reverse osmosis unit to obtain a biological extract containing hydroxytyrosol.

There is no process, using clean technologies, capable of removing, from the olive residues, a complex natural extract rich in hydroxytyrosol and containing other bioactive compounds with desirable properties, while ensuring the rejection of compounds with higher molecular weight and with biological properties. harmful.

There are commercially available, as dietary supplements, concentrates rich in hydroxytyrosol. One of these products is produced according to the patented process WO 0218310, mentioned above. It is said that this concentrate can be used in agriculture as a natural antibacterial, antiviral and antifungal agent, as well as as a therapeutic agent or food additive.

In fact, studies were carried out in order to compare the result of the process presented in Document WO

0218310, a commercial product called Hydrox®,

Creagi, and the extract obtained by the process of the present invention. Both products were tested for cytotoxicity on the CACO-2 cell line (colon

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9/25 human), and anti-proliferative activity on the HT-29 human colon cancer cell line. The results show that both extracts are non-cytotoxic to the CACO-2 cell line (human colon). However, the proliferative-inhibiting effect of the natural extract produced by the present invention on the tested human cancer cell line is superior (110% higher for polyphenol concentrations above 200 mg / L) than the biological activity exhibited by the product obtained according to the process described in WO 0218310.

In addition, the present invention uses as raw material any type of olive residue, including solid residues, and does not aim at the isolation of hydroxytyrosol itself, but rather at obtaining a complex extract rich in this compound, also comprising other bioactive compounds.

Brief description of the invention

The present invention relates to a process for obtaining a natural, bioactive concentrate, rich in hydroxytyrosol, obtained from olive residues and by-products, using clean technologies, comprising one of the following steps: (a) the supply of a liquid stream containing hydroxytyrosol and other bioactive compounds from the olive tree to an extraction column by a hydroxytyrosol fluid and a supercritical stream, in the bioactive compounds extract, or (b) which are referred to are recovered to provide a fluid stream containing hydroxytyrosol and other bioactive compounds from the olive tree to a nanofiltration unit, in which said hydroxytyrosol and other compounds

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Bioactive 10/25 are recovered in the permeate stream of said nanofiltration, which are followed by step (c), which consists of supplying the extract stream of said extraction column with supercritical fluid or the permeate stream of said nanofiltration operation to the feed compartment of a reverse osmosis unit, in which said hydroxytyrosol and bioactive compounds are retained and concentrated and concentrated in the retained fraction stream.

The natural extract comprises a minimum concentration of 15% (mass fraction) of hydroxytyrosol and a maximum concentration of 98% (mass fraction) of this compound.

The hydroxytyrosol-rich concentrate exhibits antioxidant, antimicrobial, anti-inflammatory and anticarcinogenic activities, which are superior to the activities observed for hydroxytyrosol alone, in equivalent concentration. The hydroxytyrosol-rich concentrate can be prepared as solid particles, as an aqueous solution, as an emulsion or as lipid-based nanoparticles.

The industrial application comprises the food, pharmaceutical and cosmetic industries.

Brief description of the drawings

To complement the present description, and in order to assist a better understanding of the characteristics of the invention, a detailed description of a preferred embodiment will be made, based on a set of drawings that are attached to the present specification and in which the description that the following is represented with an illustrative and not limiting character:

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11/25 figure 1 shows the chemical structure of some of the phenolic compounds present in the residues in two phases;

figure 2 shows a schematic diagram of the integrated process, which was used in the variant of carrying out the process that is the subject of the present invention;

Figure 3 shows the chromatograms obtained by liquid chromatography of: (a) a rich concentrate obtained by supercritical extraction; (b) the corresponding feeding solution. In addition to hydroxytyrosol, the presence in the extract, among others, of luteolin and hydroxycinnamic acids, such as caffeic acid and p-cumaric acid, is notorious;

Figure 4 shows a comparison between the chromatograms obtained by liquid chromatography of: (a) an aqueous extract, obtained by leaching an olive pomace with water at room temperature, which feeds the nanofiltration process, (b) the concentrate stream obtained by nanofiltration / reverse osmosis. There is a notable increase in the concentration of hydroxytyrosol and tyrosol in the current produced by nanofiltration / reverse osmosis;

Figure 5 shows the antibacterial effect of the hydroxytyrosol-rich extract obtained by integrated nanofiltration / reverse osmosis. The bacterial culture was from Ehrlichia ruminantum;

Figure 6 shows the effect of the hydroxytyrosol-rich extract obtained by integrated nanofiltration / reverse osmosis as an agent capable of reducing the proliferation of cancer cells. The cells used in this example are from the HT 29 cell line (human colonic adenocarcinoma cells).

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Detailed description of the invention

The present invention relates to a process for obtaining a natural bioactive concentrate, rich in hydroxytyrosol, from olive residues and by-products, using clean technologies.

These technologies include extraction with supercritical fluids, nanofiltration and reverse osmosis, which are used individually or in an integrated mode.

Using this process, one or more present in the olive residues plus bioactive solutes, and in the by-products, are recovered in the extract. The chemical structures of some of these compounds are shown in Figure 1.

Olive waste and by-products include: i) vegetation waters and solid waste from olive mills that work according to the three-stage process; ii) semi-solid residues from the olive mills, which work according to the two-stage process; iii) pits of olives and olive leaves.

Figure 2 shows a schematic diagram of the integrated process, which was used in the embodiment of the process that constitutes the object of the present invention. This figure comprises the following operations: extraction with biocompatible solvents, selective recovery of the solute or bioactive solutes using extraction with supercritical fluid, or nanofiltration, and reverse osmosis. The first step consists of treating the solid and semi-solid residues of the olive tree (A) by extraction with water or other biocompatible solvents (B), such as hydroalcoholic mixtures. The resulting extract can be sent directly to the extraction unit with fluid

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13/25 supercritical (I) or for the nanofiltration unit (E), or, alternatively, it can be mixed with the vegetation waters of the olive mills (C) and centrifuged (D), in order to remove the particles and other suspended solids. In the latter case, the supernatant from the centrifuge is sent to the extraction unit with supercritical fluid (I) or to the nanofiltration unit (E). The nanofiltration operation separates hydroxytyrosol and other bioactive compounds with the low molecular weight compounds, which are recovered in the permeate stream produced, from the higher molecular weight compounds, which are retained in the stream of the retained fraction. In order to increase the concentration of hydroxytyrosol and the other bioactive compounds present in the permeate, this current can be sent to a reverse osmosis unit (F). This operation produces a retained fraction stream, rich in hydroxytyrosol and other bioactive compounds of interest (G), and an aqueous stream (H), which can be reused in the extraction step (B) or discarded in the environment. The supercritical extraction process comprises a supercritical extraction column (I), where hydroxytyrosol and other bioactive compounds are recovered, and are separated from the other components of the feed stream. The current rich in hydroxytyrosol and in the other bioactive compounds can then be sent to a reverse osmosis unit (J), in order to increase its concentration.

This operation produces a stream of retained fraction rich in

hydroxytyrosol and in others compounds bioactive in interest (L), and a chain watery (M), that can to be reused in extraction step (B) or discarded at the

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14/25 environment.

In order to design a process that allows an effective recovery of bioactive compounds of interest, with high yields and high selectivity, it is necessary to define the optimal conditions for the integration of the different steps involved. This integration involves the selection of a suitable and biocompatible solvent for the extraction, and the selection of the operating conditions of the extraction unit with supercritical fluid and the steps of 10 nanofiltration and reverse osmosis.

Supercritical fluids are gases at room temperature and atmospheric pressure, or very volatile liquids, which become very dense above their critical pressure and temperature. Its properties lie between those of a gas and a liquid, resulting in a greater ability to dissolve compounds. Their relatively high specific gravity, close to the specific gravity of liquids and, simultaneously, the high diffusibility and low viscosity, similar to those of gases, allow them to extract compounds faster than conventional liquid solvents. In addition, their solvation power can be easily adjusted by varying the temperature and pressure, which makes them particularly suitable for selective fractionation of extracts.

The extraction of the target solutes from liquid raw materials, using extraction with supercritical fluids, is carried out in a column that promotes the contact between supercritical liquids and the liquid matrix. The liquid mixture is sent continuously to the top of the column by means of a pump and the liquid is fed

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Supercritical 15/25 is performed at the base of the column, by means of a compressor. The supercritical liquid and the extracted solutes, recovered from the feed liquid, leave the column via an outlet at the top. The solutes are recovered in a cyclone by expanding the supercritical fluid to 6 MPa; the supercritical fluid is compressed again and reused in the column. The pressure inside the column is controlled by a pneumatic valve located before the cyclone. This supercritical extraction column can be fed directly with vegetation waters, but it can also work with extracts previously obtained by extraction with biocompatible solvents, such as water, ethanol or mixtures of these solvents.

In the present invention, the supercritical extraction step is carried out in a temperature range between 30 ° C and 200 ° C, preferably between 30 ° C and 80 ° C, and at pressures ranging from 6 MPa to 40 MPa, preferably between 8 MPa and 20 MPa.

Any fluid or mixture of fluids in the supercritical or sub-critical liquid state can be used to carry out the process described in the present patent. The compressed fluid or mixture of fluids preferably has to be very volatile or in a gaseous state under atmospheric conditions, in order to make it easy to recover by expansion and / or evaporation after completing the extraction step. For safety reasons, the compressed fluid, or the mixture of these fluids, must be non-toxic and non-flammable and must be recyclable for later use.

Nanofiltration and reverse osmosis are techniques of

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16/25 separation through membranes for processing liquid streams, where the force responsible for transport is the difference in effective pressure between the feed compartments (therefore, the retained fraction) and the permeate. A correct selection of the nanofiltration and reverse osmosis membranes, and the operating conditions of the related equipment, allow to obtain a selective transport of the target solute or solutes and of the relevant permeation current or currents.

The nanofiltration and reverse osmosis units include a feed container, which contains the solution with the solutes to be recovered. The contents of the feed container are sent, via a centrifugal pump or a positive displacement pump, to the nanofiltration or reverse osmosis module or modules, which comprise a feed / retained fraction compartment, the membrane chosen through the which target solute or solutes are permeated, and a permeate compartment. The permeate can be removed continuously or intermittently20. After contact with the membrane, the retained fraction stream can be recycled to the supply container.

The raw material stream consists of an aqueous or hydroalcoholic solution. This solution can be water from vegetation or an extract obtained during the processing of olive residues. The target solutes to be recovered comprise all types of bioactive solutes.

The raw material stream should preferably be below 150 ° C if polymeric membranes are employed, but can be treated at

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17/25 higher temperatures if temperature-stable membranes are used, such as ceramic or metal membranes.

The membranes for nanofiltration and reverse osmosis have to act as a selective barrier in order to avoid permeation of undesirable components of the raw material, allowing the permeation of solute or solutes with a desirable bioactivity. The membranes must have characteristics that lead to a high flow rate of the target solute or solutes and a low or zero flow rate of the undesirable components of the raw material.

The membrane can be polymeric or inorganic. They can also comprise both polymeric and inorganic materials. With regard to their structure, they can also be homogeneous or composite; in the latter case, they may include several layers, consisting of different materials and / or with different morphological characteristics. Each of these layers can have a different thickness.

With regard to their chemical nature, membranes may have a hydrophobic character, meaning that they are more permeable to hydrophobic chemical species, that is, to chemical species that have an infinite activity coefficient greater than the unit in aqueous solution. Membranes can also have a hydrophilic character, which means that they are more permeable to water than to organic compounds.

The membranes can have a flat or tubular geometry and can be arranged in a plate and frame module, in a spiral wound module, in a hollow fiber module, in a

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18/25 capillary module or in a tubular module.

The raw material stream can be supplied continuously, semi-continuously or by loads. The membrane module or modules can be submerged in one or several raw material containers, or placed externally in relation to the raw material container. If more than one membrane module is used, they can be arranged in series or in parallel.

In order to establish the acting force necessary to promote the transport of the solute or solutes across the membrane, a pressure difference is established between the two compartments (feed / retained fraction and permeate) of the membrane module or modules. The value of the absolute pressure difference must be within a range of

0.5 MPa a

3.0

MPa, preferably in the

1.0 MPa a

1.5

MPa for the nanofiltration operation;

in reverse osmosis operation the range of the absolute pressure difference is between 3

MPa and 8 MPa, preferably in the range of 4 MPa to 6 MPa.

The natural concentrate rich in hydroxytyrosol (currents G and L in figure 2), obtained by the process of the present invention, contains a minimum mass fraction of 15% of hydroxytyrosol and a maximum mass fraction of 98% of this compound. The selection of operating conditions and characteristics of the nanofiltration membrane, such as its molecular weight cut-off level, allows selectively permeate bioactive compounds with the desired properties, while ensuring the retention of compounds with a higher molecular weight, which may exhibit harmful biological activity. In view of the

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19/25 selective fractionation of the bioactive compounds present in the feed stream, the molecular weight cutoff level of the nanofiltration membrane should be less than 1000 Da, preferably less than 400 Da, and most preferably less than 300 Da. The level value The molecular weight cutoff of nanofiltration membranes is calculated by determining the rejection of reference solutes, such as, galactose or sucrose, from aqueous solutions.

The concentrated extracts contain other bioactive compounds in addition to hydroxytyrosol, originally present in olives or olive leaves, according to the starting material used. The concomitant presence of hydroxytyrosol and other desired bioactive compounds, which results from the process of the present invention, creates a synergy between them which is reflected in the overall biological activity of the concentrated extracts.

As a consequence, these concentrates rich in hydroxytyrosol exhibit antioxidant, antimicrobial, anti-inflammatory and anticarcinogenic activities that are superior to the activities observed for hydroxytyrosol alone in equivalent concentration.

The hydroxytyrosol rich concentrate can be prepared in the form of a liquid, a solid or an emulsion. The liquid form refers to an aqueous solution, which can be evaporated, lyophilized or atomized, in order to produce solid particles. In addition, different specific formulations can be prepared, using biocompatible excipients and lipid matrices, in order to protect the bioactivity of the recovered compounds.

Alternatively, the hydroxytyrosol-rich concentrate

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20/25 can be prepared in the form of an emulsion, in order to make it easier to incorporate it into different products. These emulsions can be prepared using polyglycerol esters of fatty acids, glycerol esters of fatty acids, lecithin, or combinations of these emulsifiers. These formulations should have a volume fraction between 5% and 60% of hydroxytyrosol, preferably between 30% and 55%. Citric acid can be added to these emulsions in order to stabilize them.

The applications of hydroxytyrosol-rich concentrates in their various forms of supply include the food, pharmaceutical and cosmetic industries.

Examples

In order to illustrate the present invention, four examples of the use of the present invention are given. These examples are not restrictive.

Example 1:

Production of a concentrate rich in polyphenolic compounds, containing hydroxytyrosol, through an integrated process comprising extraction with biocompatible solvents and subsequent fractionation by extraction with supercritical fluid

300 g of a semi-solid residue, from a two-stage olive oil production process, were extracted with 900 mL of a hydroalcoholic solution with a volumetric ratio of 90:10 (ethanol: water). The recovered extract was centrifuged and the supernatant was used to feed an extraction column with supercritical fluid, with a structured filling provided by Sulzer; the column was 4 m high, with an internal diameter of 4 cm. The liquid

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21/25 of feed was extracted in counter-current with supercritical carbon dioxide. 500 ml of extract were recovered from the column. This extract was analyzed by liquid chromatography and the corresponding chromatogram is shown in figure 3. This figure shows the chromatograms obtained for:

(a) a rich concentrate, obtained by supercritical extraction;

(b) the corresponding feeding solution. It can be seen from figure 3 (a) that the peak corresponding to hydroxytyrosol and its derivatives represents 35% of the area of all peaks. In addition to hydroxytyrosol, the presence in the extract, among others, of luteolin and hydroxycinnamic acids with high biological activity, such as

caffeic acid and p-cumaric acid. Example 2: Production of a concentrate rich in compounds polyphenolic, containing hydroxytyrosol, using a

integrated nanofiltration / reverse osmosis process

250 mL of an aqueous extract, obtained by leaching an olive pomace with water at room temperature, was used to feed a nanofiltration unit, where it was processed at an absolute pressure difference of 1 MPa. 240 ml of permeate were obtained, with a 70% hydroxytyrosol recovery yield. The permeate obtained was subsequently treated by reverse osmosis operated at 2.5 MPa. The resulting retained fraction was characterized by liquid chromatography. Figure 4 shows the chromatograms obtained for (a) the aqueous extract that feeds the nanofiltration process, and (b) the concentrated retained fraction obtained after the described nanofiltration / reverse osmosis process. The increase

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22/25 of the hydroxytyrosol and tyrosol concentration in the current produced by nanofiltration / reverse osmosis. The nanofiltration membrane was a Desal DK, from General Electric, with a cut-off level of molecular weight of 250 Da. The 5 reverse osmosis membrane was a Filmtec SW 30 from Dow.

Example 3:

Characterization of the antimicrobial activity of a concentrated extract rich in hydroxytyrosol, produced by nanofiltration / reverse osmosis

The antimicrobial activity of a concentrated extract rich in hydroxytyrosol, obtained by nanofiltration / reverse osmosis, was evaluated by adding different concentrations of this extract to cultures of animal cells infected with the bacterium Ehrlichia ruminantum. Figure 5 15 shows that the percentage of dead bacterial cells rises up to 80% after exposure to the extract.

Example 4:

Characterization of the anticancer activity of a concentrated extract rich in hydroxytyrosol, obtained by 20 nanofiltration / reverse osmosis

The anticancer activity of a concentrated extract rich in hydroxytyrosol, obtained by integrated nanofiltration / reverse osmosis, was evaluated by adding different concentrations of this extract to cell cultures of human colonic adenocarcinoma (HT 29 cell line). Figure 6 shows that the percentage of viable cancer cells decreases to less than 20% after exposure to the extract.

References

Patents

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23/25

US 6 361 803. Cuomo et al. US 6 849 770. Fernandez-Bolanos et al. US 5 714 150. Nashman WO 0218310 ... Crea EP 1 623 960 A1. Villanova et al.

Other references

Allouche et al.,

Toward a High Yield Recovery of

Antioxidants and Purified Hydroxytyrosol from Olive Mill

Wastewaters ”,

J. Agric. Food Chem. 52 (2) (2004) 267-273

Ryan et al.,

Recovery of phenolic compounds from Olea europaea ”, Anal. Chim. Minutes 445 (2001) 67-77

DellaGreca et al.,

Low-molecular-weight components of olive oil mill wastewaters ”, Phytochem. Anal. 15 (2004)

DellaGreca et al.,

Phytotoxicity of low-molecularweight phenols from olive mill waste waters ”, Bull.

Environ. They count.

Toxicol. 67 (2001) 352-359

Drouiche et al.,

A compact process for the treatment of olive mill wastewater by combining UF and

UV / H2O2 techniques ”, Desalination 169 (2004) 81-88

Fernandez-Bolanos et al.,

Production in

Large

Quantities of

Highly Purified

Hydroxytyrosol from

LiquidSolid Waste of

Two-Phase Olive

Oil Processing or Alperujo ”,

J.

Agric. Food

Chem. 50 (2002)

6804-6811

Fernandez-Bolanos et al.,

Total Recovery of the Waste of

Two-Phase Olive Oil Processing: Isolation of Added-Value

Compounds ”, J. Agric. Food Chem. 52 (2004) 5849-5855

Lesage-Meessen et al.,

Simple phenolic content in olive oil residues as a function of extraction systems ”,

Food Chemistry 75 (2001) 501-507

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Marrugat et al., Effects of differing phenolic content in dietary olive oils on lipids and LDL oxidation. A randomized trial ”, Eur. J Nutr. 43 (2004) 140-147

Manna et al., Transport mechanism and metabolism of olive oil hydroxytyrosol in Caco-2 cells ”, FEBS Lett. 470 (2000) 341-344

Mullinacci et al., Polyphenolic content in olive oil wastewaters and related olive samples ”, J. Agric. Food Chem. 49 (2001) 3509-3514

Obied et al., Bioactivity and Analysis of Biophenols

Recovered from Olive Mill Waste, ”J. Agric. Food Chem.

(Review) 53 (2005) 823-837

Quiles et al., Olive oil phenolics: effects on DNA oxidation and redox enzyme mRNA in prostate cells ”, British 15 Journal of Nutrition 88 (2002) 225-234

Bouzid et al., Fungal enzymes as a powerful tool to release simple phenolic compounds from olive oil byproduct ”, Process Biochemistry 40 (2005) 1855-1862

Owen et al., Olive oil consumption and health: the possible role of antioxidants ”, Lancet Oncol. 1 (2000) 107112

Rodis et al., Partitioning of olive oil antioxidants between oil and water phases ”, J. Agric, Food Chem. 50 (2002) 596-601

Schieber et al., By-products of plant food processing as a source of functional compounds recent developments ”, Trends in Food Science & Technology 12 (2001) 401-413

Tuck et al., Major phenolic compounds in olive oil: metabolism and health effects ”, Journal of Nutritional 30 Biochemistry 13 (2002) 636-644

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Turano et al., An integrated ccentrifugation-ultrafiltration system in the treatment of olive mill wastewaters ”, J. Membr. Sci. 209 (2002) 519-531

Visioli et al., Antioxidant and other biological activities of olive mill wastewaters ”, J. Agric. Food Chem.

(1999) 3397-3401

Visioli et al., Olive oil phenolics are dose dependently absorbed in humans ”, FEBS Letters 468 (2000)

159-160

Visioli et al., Antioxidant and other biological activities of phenols from olives and olive oil ”, Medicinal Research Reviews 22 (2002) 65-75

Visioli et al., Biological activities and metabolical fate of olive oil phenols ”, Eur. J. Lipid Sci. Technol. 104 15 (2002) 677-684

Claims (7)

1. Process for obtaining a natural concentrate rich in natural hydroxytyrosol from olive residues and by-products, characterized by the fact that it consists of the following steps: (a) processing solid and semi-solid olive residues by extraction with water or hydroalcoholic mixtures, or (b) feeding the extract containing hydroxytyrosol and other bioactive compounds from the olive directly to a nano filtration unit with a lower molecular weight cut to 300 Da or mix the extract obtained in step (a) vegetation waters of the olive mills and centrifuge in order to remove particles and other suspended solids and feeding the supernatant obtained from the centrifuge to the said nano filtration unit, in which the said hydroxytyrosol and other low molecular weight bioactive compounds are recovered in a permeate stream from said nano filtration unit, and (c) feed the permeate stream from said nano filtration unit into the feed compartment of a reverse osmosis unit wherein said hydroxytyrosol and other bioactive compounds are retained and concentrated in a stream of f retained feed. 2. Process according to claim 1, characterized by the fact that the nano filtration unit is operated at an absolute pressure difference between the retained fraction feed compartment and the permeate compartment in the range of 0.5 MPa to 3 , 0 MPa, and where the reverse osmosis unit is operated at a Petition 870180149733, of 11/09/2018, p. 37/46 2/2 absolute pressure difference in the range of 3 MPa to 8 MPa. Process according to claim 1 or 2, characterized by the fact that nano filtration and reverse osmosis membranes are homogeneous or composite, wherein the membranes are polymeric or inorganic or both comprise polymeric and inorganic materials. Process according to any one of claims 1 to 3, characterized in that the nano filtration and the reverse osmosis membranes have a flat geometry or a tubular geometry, being arranged in a plate and frame module, in a module spiral, hollow fiber module, capillary module or tubular module. Process according to any one of claims 1 to 4, characterized by the fact that one or more membrane modules are used, arranged in series or in parallel. Process according to any one of claims 1 to 5, characterized in that at least one membrane module is submerged in one or more raw material containers, or is placed externally to the raw material container. Process according to any one of claims 1 to 6, characterized in that the raw material stream is fed to the nano filtration membrane and to the reverse osmosis unit in a continuous, semi-continuous or load mode.